rus
Issue #6/2023
A.I.Akhmetova, N.A.Nikitin, M.V.Arkhipenko, O.V.Karpova, I.V.Yaminsky
3D VISUALIZATION OF VIRIONS BY ATOMIC FORCE AND MICROLENS MICROSCOPY
3D VISUALIZATION OF VIRIONS BY ATOMIC FORCE AND MICROLENS MICROSCOPY
DOI: https://doi.org/10.22184/1993-8578.2023.16.6.338.344
Plant viruses do not have common pathogens with mammals, and therefore in recent years the development of medical and veterinary biotechnologies based on them has been actively developed. Such developments include the creation of platforms for functionally active molecules, drug delivery vehicles and biocontrast agents. The study of the structure, morphology and structural features of the surface of viral particles is an important area that makes it possible to develop new effective methods for combating viral infections and related tools of molecular medicine. In this work, the structure and properties of tobacco mosaic virus particles as a model object were studied using AFM. AFM has been demonstrated to be a suitable tool for studying viral particles.
Plant viruses do not have common pathogens with mammals, and therefore in recent years the development of medical and veterinary biotechnologies based on them has been actively developed. Such developments include the creation of platforms for functionally active molecules, drug delivery vehicles and biocontrast agents. The study of the structure, morphology and structural features of the surface of viral particles is an important area that makes it possible to develop new effective methods for combating viral infections and related tools of molecular medicine. In this work, the structure and properties of tobacco mosaic virus particles as a model object were studied using AFM. AFM has been demonstrated to be a suitable tool for studying viral particles.
Теги: bionanoscopy physics of living systems scanning probe microscopy tobacco mosaic virus virion бионаноскопия вирион вирус табачной мозаики сканирующая зондовая микроскопия физика живых систем
Original paper
3D VISUALIZATION OF VIRIONS BY ATOMIC FORCE AND MICROLENS MICROSCOPY
A.I.Akhmetova1, Cand. of Sci. (Physics and Mathematics), Junior Researcher, ORCID: 0000-0002-5115-8030
N.A.Nikitin2, Doct. of Sci. (Biology), Prof., ORCID: 0000-0001-9626-2336
M.V.Arkhipenko2, Cand. of Sci. (Biology), Senior Researcher, ORCID: 0000-0002-5575-602X
O.V.Karpova2, Doct. of Sci. (Biology), Head of Chair, ORCID: 0000-0002-0605-9033
I.V.Yaminsky1, Doct. of Sci. (Physics and Mathematics), ORCID: 0000-0001-8731-3947 / yaminsky@nanoscopy.ru
Abstract. Plant viruses do not have common pathogens with mammals, and therefore in recent years the development of medical and veterinary biotechnologies based on them has been actively developed. Such developments include the creation of platforms for functionally active molecules, drug delivery vehicles and biocontrast agents. The study of the structure, morphology and structural features of the surface of viral particles is an important area that makes it possible to develop new effective methods for combating viral infections and related tools of molecular medicine. In this work, the structure and properties of tobacco mosaic virus particles as a model object were studied using AFM. AFM has been demonstrated to be a suitable tool for studying viral particles.
Keywords: tobacco mosaic virus, virion, physics of living systems, scanning probe microscopy, bionanoscopy
For citation: A.I. Akhmetova, N.A Nikitin, M.V. Arkhipenko, O.V. Karpova, I.V. Yaminsky. 3D visualization of virions by atomic force and microlens microscopy. NANOINDUSTRY. 2023. V. 16, no. 6. PP. 338–344. https://doi.org/10.22184/1993-8578.2023.16.6.338.344.
INTRODUCTION
Tobacco mosaic virus (TMV) is a reference model for many studies, it infects plants of the solanaceae family, such as tobacco, tomato or pepper, causing characteristic mosaic patterns, especially on tobacco plants, but is harmless to mammals. TMV is an elongated hollow particle 300 nm long, 18 nm high, in which a capsid composed of 2130 capsid protein subunits forms a helix, inside which genomic single-stranded RNA is enclosed [1]. Due to its simple structure, safety for humans, stability, and accessibility, TMV is a favorite object of study for various fields of science, including molecular virology, structural biology, bioengineering, and many others.
The study of the relationship between the mechanical properties, structure, and functions of viral particles opens up ways to utilize them in a new capacity in biotechnology. Recent studies demonstrate possibility of using TMVs as microelectronic devices, vaccine components, and drug delivery systems for cancer treatment [2–6]. Atomic force microscopy is a good tool that allows 3D imaging of objects and serves as a purity and quality control of the drug for biomedical purposes. Atomic force microscopy has been used to study tobacco mosaic virus adhesion to mica and graphite, the mutual orientation of viral particles on the substrate and its possible effect on the virus, and various variants of chemical modification of the mica surface to enhance viral particles adhesion [7].
MATERIALS AND METHODS
Tobacco mosaic virus (TMV), strain U1 from the collection of the Department of Virology of Moscow State University was accumulated in tobacco plants (Nicotiana tabacum L. cv Samsun). N. tabacum plants were grown to the stage of 5–6 large leaves formation in a greenhouse with additional lighting (high-pressure sodium lamps) at 22–25 °C. Plants were infected by mechanical inoculation. A suspension of previously isolated and purified virus at a concentration of 50 µg/mL was used as the infectious material. In 1–2 weeks after infection, symptoms of systemic plant damage developed: mosaic symptoms in the form of alternating light-green and dark-green areas were observed, often accompanied by the appearance of abnormalities in the form of local swellings, clearly observed when compared with a control healthy plant. Leaves of infected plants were collected three weeks after infection, packed and frozen (–18 °C). TMV was isolated and purified by differential centrifugation as described previously [8].
Viral precipitate was dissolved in 0.01 M Tris-HCl1 pH 7.8. The TMV solution was clarified by low-speed centrifugation at 10000 g for 15 minutes. A 0.05 mL sample was taken to determine the concentration of TMV and preparation purity.
Quality of TMV isolated preparation (presence of impurities, morphology and particle size) was controlled by spectrophotometry, electrophoretic analysis and transmission electron microscopy (TEM). For analysis by transmission electron microscopy, TMV preparation was sorbed on copper grids for electron microscopy coated with collodion film additionally stabilized by carbon sputtering, negatively contrasted with 2 % uranyl acetate solution, and analyzed using a JEM-1011 electron microscope (JEOl, Japan). Size analysis was performed using ImageJ software (NIH, USA).
3D morphology of viral particles was studied using FemtoScan SPM in air on graphite and mica substrates in resonance mode, NSG10 cantilever, and image processing was performed in FemtoScan Online software [9].
RESULTS
Careful sample preparation is very important in the study of TMV by AFM. It is necessary to immobilize particles on the substrate correctly, and it is often necessary to repeatedly wash the sample, which leads to a possible change in particle geometry and its size ratio due to virus-substrate interactions [10, 11].
TEM analysis of the TMV preparation confirmed bacilliform particles presence with an average length of about 300 nm and a diameter of about 18 nm (Fig.1). No contamination of the preparation with plant or viral material of other nature was detected.
TMV concentration in purified preparation was determined using the extinction coefficient: E 260 nm 0.1 % = 3.0. The A260/A280 ratio was 1.2, which characterizes highly purified preparations of TMV and indicates that preparation consists of 95% protein and 5% RNA.
In the analysis of TMV by electrophoresis in a denaturing gradient polyacrylamide gel, one protein band corresponding to electrophoretic mobility of the TMV In the analysis of TMV by electrophoresis in a denaturing gradient polyacrylamide gel, one protein band corresponding to electrophoretic mobility of the TMV envelope protein was detected (Fig.2, track 2). No other protein bands were detected in gel stained with Coomassie G-250.
Three-dimensional images of viruses in air on graphite and on mica samples at concentration of 0.1 mg/mL were obtained by the AFM method (Fig.3 and Fig.4). Repeated washing was required to obtain good images, and its nature significantly affects the number of virus particles adsorbed on the surface.
The height of viral particles on mica and on graphite averages 16 nm ±0.8. The length of the viral particles varies from 200 nm to 2 µm because these particles can break during sample preparation and line up butt to each other.
The graphite images show not only the virus itself but also segments of destroyed viral particles, the height of the segments varying from 0.8 – 2.8±0.3 nm, and radius 22–36±3 nm (Fig.3).
It is known, that during isolation and purification of TMV, viral particles can break down to form shorter helix fragments, up to monomers of the envelope protein. In the absence of nucleic acid, the main type of aggregates formed from the envelope protein of TMV under conditions of low ionic strength of solution and neutral pH value are 20S disks (short bilayer helix fragments) of 34 subunits of the envelope protein [12]. Probably, this type of particles is observed in the AFM field of view in addition to TMV viral particles, because they are located on the surface by the end part of the helical structure.
AFM allows to identify the virus particles on substrates surface in the form of three-dimensional images. At the same time, it should be noted that the dimensions observed in AFM undergo changes. Due to interaction of the viral particle with the substrate and probe, the AFM gives a slightly underestimated value of the particle height. At the same time, the observed width of the virus particle is overestimated for the same reasons.
CONCLUSIONS
Within the framework of this work, a preparation of TMV was obtained and characterized by various methods. On the example of a model phytovirus, tobacco mosaic virus, possibility of using atomic force microscopy to study viral particles was demonstrated.
Three-dimensional images of TMV virus particles on graphite and mica substrates in the resonance mode were obtained by the AFM method, the sizes and character of particles adsorption and virus segments were obtained. The sample purity, characteristic geometric dimensions of particles, and propensity of particles to destruction depending on the substrate used were evaluated in this work.
It is shown, that particles on graphite are more prone to destruction. Nature of particle adsorption and image quality strongly depend on the washing process.
In future it is planned to carry out a detailed analysis of the surface structure of viral particles of different nature by the AFM method and to measure totality of physical and chemical properties of particles determined with nanometer spatial resolution in order to identify possibility of using viral particles in biotechnologies development: carriers, containers for functionally active molecules, vaccines.
ACKNOWLEDGMENTS
This work was supported by the MSU Development Program, project No. 23-SH04-04.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
3D VISUALIZATION OF VIRIONS BY ATOMIC FORCE AND MICROLENS MICROSCOPY
A.I.Akhmetova1, Cand. of Sci. (Physics and Mathematics), Junior Researcher, ORCID: 0000-0002-5115-8030
N.A.Nikitin2, Doct. of Sci. (Biology), Prof., ORCID: 0000-0001-9626-2336
M.V.Arkhipenko2, Cand. of Sci. (Biology), Senior Researcher, ORCID: 0000-0002-5575-602X
O.V.Karpova2, Doct. of Sci. (Biology), Head of Chair, ORCID: 0000-0002-0605-9033
I.V.Yaminsky1, Doct. of Sci. (Physics and Mathematics), ORCID: 0000-0001-8731-3947 / yaminsky@nanoscopy.ru
Abstract. Plant viruses do not have common pathogens with mammals, and therefore in recent years the development of medical and veterinary biotechnologies based on them has been actively developed. Such developments include the creation of platforms for functionally active molecules, drug delivery vehicles and biocontrast agents. The study of the structure, morphology and structural features of the surface of viral particles is an important area that makes it possible to develop new effective methods for combating viral infections and related tools of molecular medicine. In this work, the structure and properties of tobacco mosaic virus particles as a model object were studied using AFM. AFM has been demonstrated to be a suitable tool for studying viral particles.
Keywords: tobacco mosaic virus, virion, physics of living systems, scanning probe microscopy, bionanoscopy
For citation: A.I. Akhmetova, N.A Nikitin, M.V. Arkhipenko, O.V. Karpova, I.V. Yaminsky. 3D visualization of virions by atomic force and microlens microscopy. NANOINDUSTRY. 2023. V. 16, no. 6. PP. 338–344. https://doi.org/10.22184/1993-8578.2023.16.6.338.344.
INTRODUCTION
Tobacco mosaic virus (TMV) is a reference model for many studies, it infects plants of the solanaceae family, such as tobacco, tomato or pepper, causing characteristic mosaic patterns, especially on tobacco plants, but is harmless to mammals. TMV is an elongated hollow particle 300 nm long, 18 nm high, in which a capsid composed of 2130 capsid protein subunits forms a helix, inside which genomic single-stranded RNA is enclosed [1]. Due to its simple structure, safety for humans, stability, and accessibility, TMV is a favorite object of study for various fields of science, including molecular virology, structural biology, bioengineering, and many others.
The study of the relationship between the mechanical properties, structure, and functions of viral particles opens up ways to utilize them in a new capacity in biotechnology. Recent studies demonstrate possibility of using TMVs as microelectronic devices, vaccine components, and drug delivery systems for cancer treatment [2–6]. Atomic force microscopy is a good tool that allows 3D imaging of objects and serves as a purity and quality control of the drug for biomedical purposes. Atomic force microscopy has been used to study tobacco mosaic virus adhesion to mica and graphite, the mutual orientation of viral particles on the substrate and its possible effect on the virus, and various variants of chemical modification of the mica surface to enhance viral particles adhesion [7].
MATERIALS AND METHODS
Tobacco mosaic virus (TMV), strain U1 from the collection of the Department of Virology of Moscow State University was accumulated in tobacco plants (Nicotiana tabacum L. cv Samsun). N. tabacum plants were grown to the stage of 5–6 large leaves formation in a greenhouse with additional lighting (high-pressure sodium lamps) at 22–25 °C. Plants were infected by mechanical inoculation. A suspension of previously isolated and purified virus at a concentration of 50 µg/mL was used as the infectious material. In 1–2 weeks after infection, symptoms of systemic plant damage developed: mosaic symptoms in the form of alternating light-green and dark-green areas were observed, often accompanied by the appearance of abnormalities in the form of local swellings, clearly observed when compared with a control healthy plant. Leaves of infected plants were collected three weeks after infection, packed and frozen (–18 °C). TMV was isolated and purified by differential centrifugation as described previously [8].
Viral precipitate was dissolved in 0.01 M Tris-HCl1 pH 7.8. The TMV solution was clarified by low-speed centrifugation at 10000 g for 15 minutes. A 0.05 mL sample was taken to determine the concentration of TMV and preparation purity.
Quality of TMV isolated preparation (presence of impurities, morphology and particle size) was controlled by spectrophotometry, electrophoretic analysis and transmission electron microscopy (TEM). For analysis by transmission electron microscopy, TMV preparation was sorbed on copper grids for electron microscopy coated with collodion film additionally stabilized by carbon sputtering, negatively contrasted with 2 % uranyl acetate solution, and analyzed using a JEM-1011 electron microscope (JEOl, Japan). Size analysis was performed using ImageJ software (NIH, USA).
3D morphology of viral particles was studied using FemtoScan SPM in air on graphite and mica substrates in resonance mode, NSG10 cantilever, and image processing was performed in FemtoScan Online software [9].
RESULTS
Careful sample preparation is very important in the study of TMV by AFM. It is necessary to immobilize particles on the substrate correctly, and it is often necessary to repeatedly wash the sample, which leads to a possible change in particle geometry and its size ratio due to virus-substrate interactions [10, 11].
TEM analysis of the TMV preparation confirmed bacilliform particles presence with an average length of about 300 nm and a diameter of about 18 nm (Fig.1). No contamination of the preparation with plant or viral material of other nature was detected.
TMV concentration in purified preparation was determined using the extinction coefficient: E 260 nm 0.1 % = 3.0. The A260/A280 ratio was 1.2, which characterizes highly purified preparations of TMV and indicates that preparation consists of 95% protein and 5% RNA.
In the analysis of TMV by electrophoresis in a denaturing gradient polyacrylamide gel, one protein band corresponding to electrophoretic mobility of the TMV In the analysis of TMV by electrophoresis in a denaturing gradient polyacrylamide gel, one protein band corresponding to electrophoretic mobility of the TMV envelope protein was detected (Fig.2, track 2). No other protein bands were detected in gel stained with Coomassie G-250.
Three-dimensional images of viruses in air on graphite and on mica samples at concentration of 0.1 mg/mL were obtained by the AFM method (Fig.3 and Fig.4). Repeated washing was required to obtain good images, and its nature significantly affects the number of virus particles adsorbed on the surface.
The height of viral particles on mica and on graphite averages 16 nm ±0.8. The length of the viral particles varies from 200 nm to 2 µm because these particles can break during sample preparation and line up butt to each other.
The graphite images show not only the virus itself but also segments of destroyed viral particles, the height of the segments varying from 0.8 – 2.8±0.3 nm, and radius 22–36±3 nm (Fig.3).
It is known, that during isolation and purification of TMV, viral particles can break down to form shorter helix fragments, up to monomers of the envelope protein. In the absence of nucleic acid, the main type of aggregates formed from the envelope protein of TMV under conditions of low ionic strength of solution and neutral pH value are 20S disks (short bilayer helix fragments) of 34 subunits of the envelope protein [12]. Probably, this type of particles is observed in the AFM field of view in addition to TMV viral particles, because they are located on the surface by the end part of the helical structure.
AFM allows to identify the virus particles on substrates surface in the form of three-dimensional images. At the same time, it should be noted that the dimensions observed in AFM undergo changes. Due to interaction of the viral particle with the substrate and probe, the AFM gives a slightly underestimated value of the particle height. At the same time, the observed width of the virus particle is overestimated for the same reasons.
CONCLUSIONS
Within the framework of this work, a preparation of TMV was obtained and characterized by various methods. On the example of a model phytovirus, tobacco mosaic virus, possibility of using atomic force microscopy to study viral particles was demonstrated.
Three-dimensional images of TMV virus particles on graphite and mica substrates in the resonance mode were obtained by the AFM method, the sizes and character of particles adsorption and virus segments were obtained. The sample purity, characteristic geometric dimensions of particles, and propensity of particles to destruction depending on the substrate used were evaluated in this work.
It is shown, that particles on graphite are more prone to destruction. Nature of particle adsorption and image quality strongly depend on the washing process.
In future it is planned to carry out a detailed analysis of the surface structure of viral particles of different nature by the AFM method and to measure totality of physical and chemical properties of particles determined with nanometer spatial resolution in order to identify possibility of using viral particles in biotechnologies development: carriers, containers for functionally active molecules, vaccines.
ACKNOWLEDGMENTS
This work was supported by the MSU Development Program, project No. 23-SH04-04.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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